The present invention relates to high-throughput systems for analyzing samples by both liquid chromatography and mass spectrometry.
Mass spectrometry (MS) is an important analysis technique in many industrial and academic fields. MS exploits the behavior of the gas-phase ions (i.e., gaseous molecules with a non-zero charge) of a molecule in response to applied electric and magnetic fields in order to deduce the composition of the molecule. The ionization process breaks a molecule into its components, the mass of each of which is then determined by analyzing the trajectory of the components as they travel through the mass spectrometer. Knowing the mass and composition of a desired molecule is especially important for pharmaceutical research, particularly in the synthesis of novel and uncharacterized molecules.
The ability to identify molecules using MS complements another analytical technique, high performance liquid chromatography (HPLC). HPLC is a physical method of separation involving sample dissolved in one or more solvents (or a xe2x80x9cmobile phasexe2x80x9d), wherein the solution is forced through a separation medium, typically a column packed with a fine particulate matter (or a xe2x80x9cstationary phasexe2x80x9d). The stationary phase, through chemical affinities, friction and/or hydrodynamic effects, acts to separate the compound (typically a complex mixture of molecules of varying size and mass) into its individual components or species. Following separation in a HPLC column, the output stream contains a series of regions having an elevated concentration of an individual component or xe2x80x9cspeciesxe2x80x9d of the sample. Each of these regions appear on a chromatogram as a concentration xe2x80x9cpeak,xe2x80x9d and sometimes even comprise visible bands within the output stream. Thus, HPLC acts to provide relatively pure and discrete samples of each of the components of a compound. However, it is difficult to identify or characterize individual components using only HPLC, particularly when novel or previously uncharacterized compounds are used.
By coupling the output of an HPLC system to a MS system, it becomes possible to accurately identify and characterize each band, i.e., the component molecules of a compound. Although several techniques may be used to introduce the output of an HPLC apparatus into a MS instrument, one prevalent technique is to use an electrospray (ES) interface. ES is a xe2x80x9csoftxe2x80x9d ionization technique. That is, ES does not rely on extremely high temperatures or extremely high voltages to accomplish ionization, which is advantageous for the analysis of large, complex molecules that tend to decompose under harsh conditions. ES uses the combination of an applied electric field and compressed gas to generate charged droplets of the sample solution. The application of drying gases in conjunction with a vacuum causes the droplets to grow increasingly smaller until a desolvated, charged sample molecule is produced.
Recent advances in chromatography have resulted in the development of high throughput liquid chromatography (HTLC), which employs improved chromatographic columns to dramatically reduce the amount of time needed to perform high-resolution separations. Also, demand for increased throughput capacity has led to the development of multiplexed chromatography systems, which include multiple separation columns to permit the separation of multiple chemical mixtures in parallel.
Typically, a single MS instrument has a single ES component (or other suitable ionization/input device) and, as such, may only accommodate the output of a single HPLC column. Moreover, ES/MS instruments are extremely complex and expensive to operate and maintain. Thus, it would be advantageous to have the ability to couple a multiplexed HTLC system to a single ES/MS instrument, thereby minimizing the number of ES/MS systems required to analyze multiple output streams. Several systems for multiplexing multiple separation columns into a single ES/MS system have been proposed. For example, U.S. Pat. No. 6,410,915 to Bateman et al.; U.S. Pat. No. 6,191,418 to Hindsgaul et al.; U.S. Pat. No. 6,066,848 to Kassel et al.; and U.S. Pat. No. 5,872,010 to Karger et al. each show some variation of a multiplexed HPLC/MS system where the outputs of multiple simultaneously-operated separation columns are periodically sampled by a single MS device. However, in such real-time multiplexed HPLC/MS systems, only one separation column output stream can be sampled at a given time. While one stream is being analyzed, the others must continue to flow, as these systems have no storage capacity. This inherently results in data loss. To mitigate this data loss, sampling must occur very quickly. The MS instrument thus receives very small plugs of sample, reducing the ability of the instrument to integrate data in order to eliminate noise. Signal clarity, in turn, suffers.
Optical detection devices, such as UV-Visible spectrum detectors that are capable of collecting data in parallel, may be used to selectively control the sampling of multiple separation column output streams so that a region of interest in a particular stream may be sampled more frequently, thereby ameliorating the limitations of a sampling system. However, if two or more regions of interest coincide in time, which frequently occurs, such pre-screening-based sample selection will be of limited value.
U.S. Pat. No. 6,318,157 to Corso et al. (xe2x80x9cCorsoxe2x80x9d) describes a multiplexed HPLC/MS device where gradient separations are performed by staggering the initiation, of separations in four separate columns by using input lines of varying length. In this manner, each output stream may be analyzed continuously by the MS instrument. The staggering technique taught by Corso effectively acts as four serial separations. While some efficiencies are gained by not having to prepare a single column four times, the overall run time of the four columns run in a stagger is much longer than the run time of four columns run simultaneously. Also, this system incorporates numerous rotary valves that may create dead volumes and induce error. Moreover, the necessary amount of stagger (i.e., the length of each input line) must be calculated in advance to insure that regions of interest have no temporal overlap, which may be difficult when characterizing unknown compounds. Corso also suggests that the staggering of inputs is not necessary for isocratic separations; however, Corso does not indicate how overlap of regions of interest can be avoided. Presumably, a sampling technique is used, thus creating the same data loss and signal clarity issues discussed above.
Accordingly, there exists a need for a multiplexed HPLC/MS system that permits the substantially continuous analysis of the output streams of multiple simultaneously-operated separation columns with minimal loss of data and/or signal clarity.